Fig 2: Depletion of heat shock factor 1 (HSF1) increases estrogen receptor α (ERα) levels and transcriptional activation of ERα-regulated genes.(A) ERα-activated genes are increased after HSF1 depletion in LCC9 cells. Total mRNA levels were measured using qRT-PCR. Data represent the log2 fold change between LCC9-sgCtrl and sgHSF1. Means ± SEM (n = 5–7 biological replicates) are presented and P-values were calculated using a bivariate t test. (B) Increased expression of ERα in HSF1-depleted LCC9 cells. Western blot analysis of total protein extracts showing a 4.7-fold increase in the ERα expression, whereas transcriptional coregulators BRD4 (0.9-fold) and MED23 (1.0-fold) are not affected when comparing LCC9-sgCtrl and sgHSF1 cells. Protein quantifications were performed using the ImageJ software and GAPDH as a loading control. (C, D, E) Recruitment of (C) HSF1, (D) MED1, and (E) BRD4 are decreased at HSF1-activated genes in LCC9-sgHSF1 compared with sgCtrl. Recruitment was measured by ChIP-qPCR using the promoters of HSPH1, HSP90AA1, and HSPD1 which were all described to be occupied and regulated by HSF1 (Mahat et al, 2016). Percentage of input means ± SEM (n = 4–7 biological replicates) are represented and P-values were calculated using a bivariate t test. (F, G, H) Recruitment of (F) ERα, (G) MED1, and (H) BRD4 are increased at ERα-activated genes in LCC9-sgHSF1 compared with sgCtrl. Recruitment was measured by ChIP-qPCR at ERα-controlled noncoding regulatory regions: the promoter of TFF1, the enhancer of GREB1, and the enhancer of ESR1 (Fournier et al, 2016). Percentage of input means ± SEM (n = 3–7 biological replicates) are represented and P-values were calculated using a bivariate t test. *P ≤ 0.05.

Fig 3: Derepressed IAPs form nuclear foci that associate with RNAPII condensates and incorporate nearby genes.a, Representative images of individual z-slices (same z) of RNA-FISH and RNAPII IF signal, and an image of the merged channels. The nuclear periphery determined by DAPI staining is highlighted as a white contour. The zoom column displays the region of the images highlighted in a yellow box (enlarged for greater detail). Merge of the nuclear z-projections is displayed, and overlapping pixels between the RNA-FISH and IF channels are highlighted in white. Displayed MOC and Pearson’s correlation coefficient (r) values are an average obtained from 24 analyzed nuclei. Scale bars, 2.5 μm. b, Same as a, except with MED1 IF. c, Distance of IAP RNA-FISH foci to the nearest RNAPII or MED1 IF puncta. Each dot represents one IAP RNA-FISH focus. d, Meta representations of RNAPII ChIP with reference exogenous genome (ChIP-RX) (left) and MED23 ChIP–seq (right) read densities at IAP, MMERVK and MMETn ERVs in control (DMSO-) and dTAG-13 (24 h)-treated mESCs. The mean read densities are displayed ±2 kb around the indicated elements. The genomic elements were length normalized. e, Genome browser tracks at the Cthrc1 locus. Note the independent transcription initiation events at Cthrc1 and MMETn, ruling out that the MMETn acts as an alternative Cthrc1 promoter. Rpm, reads per million. f, Representative images of individual z-slices (same z) of RNA-FISH and IF signal, and an image of the merged channels. The nuclear periphery determined by DAPI staining is highlighted as a white contour (scale bars, 2.5 μm). Also shown are averaged signals of either RNA-FISH or IF centered on the Cthrc1 FISH foci or randomly selected nuclear positions (scale bars, 0.5 μm). r denotes a Spearman’s correlation coefficient. g, Same as f, except with NFYA IF. h, qRT–PCR data for IAP RNA, Cthrc1 mRNA and the Pri-miR-290-295 transcript in control and ERV-triple knockout (TKO) cells. Data are presented as mean values ± s.d. from six biological replicates. P values are from two-tailed t-tests. ****P < 1 × 10−4. i, Principal component (PC) plot of Hi-C interactions at an ERV-rich locus on chromosome 12. j, Pile-up analysis of contacts between IAPs, MMERVKs, MMETns and transcribed genes in wild-type and TRIM28-degraded mESCs.

Fig 4: Condensate hijacking additional data.a, b. Co-localization between the IAP RNA and (a) RNAPII puncta and (b) MED23 puncta in TRIM28-degraded mESCs. Displayed are separate images of the RNA-FISH and IF signal, and an image of the merged channels. The nuclear periphery determined by DAPI staining is highlighted as a white contour. The zoom column displays the region of the images highlighted in a yellow box zoomed in for greater detail. After 24 h dTAG-13 treatment, small nuclear puncta appear, and after 48 h of dTAG-13 treatment, large nuclear foci are visible. Scale bars: 2.5 μm. c. Scheme of the 1-6 hexanediol (1-6 HD) treatment experiments. d. Representative images of RNAPII immunofluorescence in control and 1-6 HD-treated cells. 1-6 HD partially dissolved the punctate localization of RNAPII. Scale bars: 5 μm. e. Transcription of the nascent Cthcr1 RNA is reduced by 30 min 1% 1-6 hexanediol-treatment in TRIM28-degraded cells. The bar plots show qRT-PCR data as fold change normalized to the DMSO control across 6 and 3 biological replicates for 24 h and 48 h timepoints, respectively. Note that the IAP RNA does not contain introns; thus, the IAP RNA qRT-PCR detects the steady state pool of IAP RNAs. Each dot represents a data point, and bar indicates the mean. P values are from two-tailed t tests. NS: not significant.

Fig 5: MED13L bridges the CDK8 kinase module with the core Mediator. (A, B) Immunoprecipitation (IP) experiments using antibodies to MED13L, MED6, MED8, MED1, MED4, MED23, MED12, CCNC and CDK8 in PC9 and A549 cells. (C) A carton model indicating the role of MED13L as a vital linker between the core Mediator and the kinase module. (D) IP experiments between P300, MED13L and MED12 in NSCLC cells.